The serotonin transporter (SERT) on the platelet surface is the major mechanism for the uptake of plasma serotonin (5-hydroxytryptamine; 5HT), which subsequently is stored in platelet dense granules. The 5HT uptake capacity of the platelets depends on the number of SERT molecules on the plasma membrane. Earlier studies established that surface SERT expression in platelets shows a biphasic relationship to extracellular 5HT concentrations. Specifically, in platelets, plasma membrane SERT levels and 5HT uptake by SERT initially rise as plasma 5HT levels are increased, but then fall below normal as the plasma 5HT level continues to rise. Indeed, our in vivo and in vitro studies confirm a dynamic relationship between extracellular 5HT elevations, loss of surface SERT, and depletion of platelet 5HT. Additionally, we show for the first time that 5HT-depleted platelets appear to aggregate more readily, and elevating plasma 5HT in vivo appears to trigger platelet 5HT depletion and platelet aggregation. This project by a new investigator is designed to define the biochemical mechanisms by which elevated 5HT down-regulates the expression of SERT on the platelet membrane, and to examine the impact of this abnormality on platelet aggregation. The latter findings represent the future direction of the P.I., who is a protein biochemist transitioning to physiological models. The preliminary studies support the hypothesis that high extracellular 5HT leads to abnormalities in the platelet trafficking of SERT, which reduces the density of SERT molecules on the plasma membrane to deplete 5HT content. These events appear to promote platelet aggregation, a new finding just uncovered. Mechanistically, our studies will investigate the link between elevated extracellular 5HT and loss of surface SERT by focusing on the intracellular tethering of SERT by Rab4, and the 5HT-mediated phosphorylation of vimentin that promotes SERT internalization in platelets. Two model systems will be used: (a) 5HT-pretreated platelets isolated from untreated C57BL/6J mice, and (b) platelets isolated from C57BL/6J mice in which plasma 5HT will be elevated for 24 hours by osmotic minipump to ensure that our finding of SERT down-regulation by high extracellular 5HT in isolated platelets occurs in vivo and impacts platelet function. Overall, this project will provide the first detailed information on the 5HT-mediated biochemical pathways that regulate the number of functional SERT molecules on the platelet surface. The importance of understanding the structure, function, and regulation of SERT is underscored by the observations that plasma 5HT may be elevated in the plasma, either locally or globally, during atherosclerosis, hypertension, stroke, and other cardiovascular diseases.